Particle and pharmaceutical composition comprising an insoluble camptothecin compound with double coreshell structure and method for manufacturing the same

ABSTRACT

A a drug delivery system having an inner core-shell like structure containing a poorly soluble camptothecin compound and a water-soluble camptothecin compound, and an outer amphiphilic polymer shell surrounding the inner core-shell like structure, a manufacturing method therefor, and uses of the drug delivery system in treating cancer are disclosed. The core-shell structured particles form very stable particles and show a mono-distribution of particles before and after freeze-drying. The particles show excellent results compared with existing particles which do not contain the inner core-shell like structure, in animal efficacy tests and pharmacokinetic tests.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional application of U.S. patent applicationSer. No. 16/015,470 filed Jun. 22, 2018 (allowed), which claims priorityto and the benefit of Korean Patent Application No. 10-2017-0079354filed in the Korean Intellectual Property Office on 22 Jun. 2017, thedisclosures of which are incorporated herein by reference.

TECHNICAL FIELD

The present disclosure relates to a drug delivery system having a doublecore-shell structure and, specifically, to a nano-drug delivery systemhaving an inner core containing a poorly soluble camptothecin compoundand a water-soluble camptothecin compound and an amphiphilic polymershell, and to a manufacturing method therefor. Uses of the drug deliverysystem in treating cancer are disclosed.

BACKGROUND ART

A solubilizing step is essentially needed to develop a poorly solubledrug to be used for injection, and in order to overcome such a problem,emulsions containing surfactants, micro-emulsions, liposomes, micelles,pegylation, or making prodrugs, have been employed. 7-Ethyl 10-hydroxycamptothecin (SN-38), which is one of the drugs having strongestactivity as camptothecin-based anticancer drugs, is supplied asirinotecan in a prodrug form (trade name: CAMTOSAR®, manufactured byNovartis), which is mainly hydrolyzed by carboxylesterase II (CESII) invivo to be converted into SN-38 in an active form. However, the rate ofconversion is as very low as 2-8% and the deviation thereof is also verylarge, and thus such a drug has difficulty in proper administration tocancer patients in need of precise dosage adjustment, with the resultthat the effects and side effects thereof are difficult to predict.

Another method for solubilization of a poorly soluble drug is tomicellize a drug to have a nano-sized particle diameter. In order toallow a nanomicelle injection to secure sterility and stability, ananomicelle aqueous solution is sterile-filtered through a 0.22-μmfilter, and then powdered into solid through freeze-drying, during themanufacturing process. In this process, when a freeze-dried product isreconstituted for injection by dissolving the product in a solvent(saline or the like), a large amount of macroparticles (200 nm or moreto several tens of μm) may be generated due to the agglomeration ofmicelles themselves. In particular, particles larger than 5 μm may causesevere side effects when injected into the human body, and thus arestrictly managed through insoluble particulate matter tests in USPharmacopoeia, European Pharmacopoeia, and Korean Pharmacopoeia.Therefore, a solubilizing method whereby the particle sizes are littlechanged before and after freeze-drying in spite of micellization,particles show a mono-distribution, and especially, there are noparticles of several μm or more.

Meanwhile, it is essential to dissolve an active ingredient in anappropriate solvent for an injection, but camptothecin or SN-38, whichis a hydrophobic camptothecin-based compound, is hardly dissolved inwater or most volatile polar organic solvents (methanol, ethanol,acetonitrile, ethyl acetate, etc.) used in pharmaceutical formulation.Solvents capable of dissolving such the hydrophobic camptothecin-basedcompounds are limited to non-volatile solvents, such as dimethylsulfoxide (DMSO), dimethyl formamide, toluene, and dioxane. However,these non-volatile solvents are toxic and, thus, required to be removedby complicate process such as a dialysis procedure. However, it isdifficult to completely remove the non-volatile solvents, and residualsolvents could cause side effects.

Therefore, the present inventors searched and endeavored to developstable particle compositions of severely poorly soluble camptothecincompounds, which are free of the above problems and can be directlyadministered in an active form but not a prodrug form. Furthermore,there was a need for a severely poorly soluble camptothecin compoundsnanoparticle formulation which does not undergo significant particlesize changes before and after freeze-drying, and free ofmacro-agglomerated particles of several μm or more.

Throughout the entire specification, many patent documents arereferenced and their citations are represented. The disclosures of citedpapers and patent documents are entirely incorporated by reference intothe present specification, and the level of the technical field withinwhich the present invention falls and details of the embodiments areexplained more clearly.

SUMMARY

An aspect of the present invention is to provide a particle including:i) a hydrophobic camptothecin-based compound; ii) a hydrophiliccamptothecin-based compound; and iii) an amphiphilic block copolymercomposed of a hydrophobic block and a hydrophilic block.

Another aspect of the present invention is to provide a pharmaceuticalcomposition for treating cancer containing the particle and apharmaceutically acceptable carrier.

Still another aspect of the present invention is to provide a method formanufacturing a particle, the method including:

-   -   (a) forming an inner core-shell containing a hydrophobic        camptothecin-based compound and a hydrophilic camptothecin-based        compound; and (b) forming an outer core-shell containing an        amphiphilic copolymer.

According to another aspect, a method of treating cancer in a subject inneed thereof which comprises administering an effective amount of thepharmaceutical composition to the subject.

Other purposes and advantages of the present disclosure will become moreobvious with the following detailed description of the invention,claims, and drawings.

In accordance with an aspect of the present invention, there is providedEmbodiments 1 to 35 below:

1. A particle comprising:

-   -   i) a hydrophobic camptothecin-based compound;    -   ii) a hydrophilic camptothecin-based compound; and    -   iii) an amphiphilic block copolymer composed of a hydrophobic        block and a hydrophilic block.

2. The particle of Embodiment 1, wherein the hydrophobiccamptothecin-based compound is at least one selected from the groupconsisting of 7-ethyl-10-hydroxycamptothecin (SN-38), camptothecin,10-hydroxycamptothecin, and a pharmaceutical acceptable salt thereof.

3. The particle of Embodiment 1, wherein the hydrophiliccamptothecin-based compound is at least one selected from irinotecan,topotecan, belotecan, exatecan, lurtotecan, sinotecan, rubitecan,9-nitrocamptothecin, 9-aminocamptothecin, gimatecan, BNP-1530, DB-67,BN-80915, BN-80927, a pharmaceutically acceptable salt thereof, aglucuronide metabolite thereof, and a glucuronide metabolite of thehydrophobic camptothecin-based compound.

4. The particle of Embodiment 1, wherein the amphiphilic block copolymeris composed of A-B or A-B-A blocks,

-   -   (a) wherein A is a hydrophilic polymer, which is monomethoxy        polyethylene glycol, dimethoxy polyethylene glycol, polyethylene        glycol, polypropylene glycol, monomethoxy polypropylene glycol,        polyethylene oxide, polyacrylic acid, or a polymer thereof; and    -   (b) wherein B is a hydrophobic polymer, which is polylactic        acid, polylactide, polyglycolic acid, polyglycolide, a        polylactic acid-co-glycolic acid copolymer, polymandelic acid,        polycaprolactone, polydioxan-2-one, polyglutamic acid,        polyaspartic acid, polyornithine, polyorthoester, a derivative        thereof, or a copolymer of two or more compounds selected        therefrom.

5. The particle of Embodiment 4, wherein the number average molecularweight of the hydrophilic polymer A is 500-10,000 Da.

6. The particle of Embodiment 4, wherein the number average molecularweight of the hydrophobic polymer B is 500-10,000 Da.

7. The particle of Embodiment 1, wherein the weight ratio of thehydrophobic camptothecin-based compound and the hydrophiliccamptothecin-based compound is 1:10 to 10:1.

8. The particle of Embodiment 1, wherein the weight ratio of the sum ofthe hydrophobic camptothecin-based compound and the hydrophiliccamptothecin-based compound and the amphiphilic block copolymer is 1:200to 10:1.

9. The particle of Embodiment 1, wherein the number average particlesize of the particle is 10-500 nm.

10. The particle of Embodiment 1, wherein the particle has a doublecore-shell structure:

-   -   (a) an inner core-shell containing the hydrophilic        camptothecin-based compound and the hydrophobic        camptothecin-based compound; and    -   (b) an outer core-shell containing the amphiphilic block        copolymer.

11. The particle of Embodiment 1, wherein the particle has a bilayermicelle structure.

12. A pharmaceutical composition for treating cancer, comprising theparticle of any one of Embodiments 1 to 11 and a pharmaceuticallyacceptable carrier.

13. The composition of Embodiment 12, wherein the cancer is selectedfrom the group consisting of gastric cancer, ovarian cancer, uterinecancer, small cell lung cancer, non-small cell lung cancer, pancreaticcancer, breast cancer, esophageal cancer, oral cancer, rectal cancer,colon cancer, large intestine cancer, kidney cancer, prostate cancer,melanoma, liver cancer, gall bladder and other biliary tract cancer,thyroid cancer, bladder cancer, brain and central nervous system cancer,bone cancer, skin cancer, non-Hodgkin's and Hodgkin's lymphoma, andblood cancer.

14. The composition of Embodiment 12, wherein the composition furthercomprises different types of anticancer drugs.

15. The composition of claim 12, further comprises sucrose, mannitol,sorbitol, glycerin, trehalose, and a polyethylene glycol excipient, anda cyclodextrin excipient.

16. A method for treating cancer in a subject, the method comprisingadministering an effective amount of the pharmaceutical composition ofany one of Embodiments 1 to 15 to the subject.

17. The method of Embodiment 16, wherein the subject is human, mouse,rat, guinea pig, dog, cat, horse, cow, pig, monkey, chimpanzee, baboon,or rhesus monkey.

18. A method for manufacturing the particle of Embodiment 1, the methodcomprising:

-   -   (a) forming an inner core-shell containing a hydrophobic        camptothecin-based compound and a hydrophilic camptothecin-based        compound; and    -   (b) forming an outer core-shell containing an amphiphilic        copolymer.

19. The method of Embodiment 18, wherein step (a) comprises mixing thehydrophobic camptothecin-based compound and a hydrophiliccamptothecin-based compound in an organic solvent; and wherein step (b)comprises mixing the inner core-shell and the amphiphilic blockcopolymer in an aqueous solvent.

20. The method of Embodiment 18, wherein step (a) comprises mixing abasic aqueous solution, in which a hydrophobic camptothecin compound isdissolved, and an aqueous solution, in which a hydrophilic camptothecincompound is dissolved; and wherein step (b) comprises mixing the innercore-shell and the amphiphilic block copolymer in an aqueous solvent.

21. The method of Embodiment 20, wherein the aqueous solution in whichthe hydrophilic camptothecin compound is dissolved is a basic, neutral,or acidic aqueous solution.

22. The method of Embodiment 20, wherein step (a) comprises:

-   -   (a1) mixing a basic aqueous solution, in which a hydrophobic        camptothecin-based compound is dissolved, and a basic, neutral,        or acidic aqueous solution, in which a hydrophilic        camptothecin-based compound is dissolved; and    -   (a2) lowering the pH of the mixed aqueous solution to 7 or        lower.

23. The method of Embodiment 18, wherein step (a) comprises mixing thehydrophobic camptothecin-based compound and the hydrophiliccamptothecin-based compound in an organic solvent; and

-   -   wherein step (b) comprises mixing a mixture of the hydrophobic        camptothecin-based compound and the hydrophilic        camptothecin-based compound with the amphiphilic block copolymer        in an organic solvent.

24. The method of any one of Embodiments 18 to 23, wherein thehydrophobic camptothecin-based compound is at least one selected fromthe group consisting of 7-ethyl-10-hydroxycamptothecin (SN-38),camptothecin, 10-hydroxycamptothecin, and a pharmaceutical acceptablesalt thereof.

25. The method of any one of Embodiments 18 to 24, wherein thehydrophilic camptothecin-based compound is at least one selected fromirinotecan, topotecan, belotecan, exatecan, lurtotecan, sinotecan,rubitecan, 9-nitrocamptothecin, 9-aminocamptothecin, gimatecan,BNP-1530, DB-67, BN-80915, BN-80927, a pharmaceutically acceptable saltthereof, a glucuronide metabolite thereof, and a glucuronide metaboliteof the hydrophobic camptothecin-based compound.

26. The method of any one of Embodiments 18 to 24, wherein theamphiphilic block copolymer is composed of A-B or A-B-A blocks,

-   -   (a) wherein A is a hydrophilic polymer, which is monomethoxy        polyethylene glycol, dimethoxy polyethylene glycol, polyethylene        glycol, polypropylene glycol, monomethoxy polypropylene glycol,        polyethylene oxide, polyacrylic acid, or a polymer thereof; and    -   (b) wherein B is a hydrophobic polymer, which is polylactic        acid, polylactide, polyglycolic acid, polyglycolide, a        polylactic acid-co-glycolic acid copolymer, polymandelic acid,        polycaprolactone, polydioxan-2-one, polyglutamic acid,        polyaspartic acid, polyornithine, polyorthoester, a derivative        thereof, or a copolymer of two or more compounds selected        therefrom.

27. The method of Embodiment 26, wherein the molecular weight of thehydrophilic polymer A is 500-10,000 Da.

28. The method of Embodiment 26, wherein the molecular weight of thehydrophobic polymer B is 500-10,000 Da.

29. The method of any one of Embodiments 18 to 24, wherein the weightratio of the hydrophobic camptothecin-based compound and the hydrophiliccamptothecin-based compound is 1:10 to 10:1.

30. The method of any one of Embodiments 18 to 24, wherein the weightratio of the sum of the hydrophobic camptothecin-based compound and thehydrophilic camptothecin-based compound and the amphiphilic blockcopolymer is 1:200 to 10:1.

31. The method of any one of Embodiments 18 to 24, wherein the organicsolvent is a C1-C5 alcohol (methanol, ethanol, propanol, butanol,n-butanol, iso-propanol, 1-pentanol, 2-butoxyethanol, isobutyl alcohol,and etc), an alkyl acetate, acetone, acetonitrile, chloroform, benzene,toluene, xylene, acetone, fluoroalkane, pentane, hexane,2,2,4-trimethylpentane, decane, cyclohexane, cyclopentane,diisobutylene, 1-pentene, 1-chlorobutane, 1-chloropentane, diisopropylether, 2-chloropropane, 1-chloropropane, chlorobenzene, benzene, diethylether, diethyl sulfide, dichloromethane, 1,2-dichloroethane, aniline,diethyl amine, an ether, carbon tetrachloride, tetrahydrofuran (THF), ora mixed solvent thereof.

32. The method of Embodiment 21 or 22, wherein the acidic aqueoussolution includes at least one selected from pharmaceutically acceptableinorganic acids including hydrochloric acid, nitric acid, sulfuric acid,and phosphoric acid, or organic acids including citric acid, malic acid,lactic acid, acetic acid, and tartaric acid.

33. The method of Embodiment 21 or 22, wherein a pH of the acidicaqueous solution is 1.0 to 6.

34. The method of Embodiment 20, wherein the basic solution includes atleast one selected from the group consisting of an inorganic alkali, analkali salt of an organic acid, and an alkyl amine, the inorganic alkaliincluding sodium hydroxide, potassium hydroxide, sodiumdihydrogenphosphate, potassium dihydrogenphosphate, magnesium hydroxide,sodium carbonate, and sodium hydrogencarbonate.

35. The method of Embodiment 20, wherein a pH of the basic aqueoussolution is 8 to 13.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1a and 1b are graphs showing the dynamic light scattering (DLS)measurement results of the size distributions of particles in an aqueoussolution after freeze-drying, with respect to comparative core-shellparticles (monolayer micelles) composed of SN-38 and irinotecan.

FIG. 1c is a graph showing the dynamic light scattering (DLS)measurement results of the size distributions of particles in an aqueoussolution after freeze-drying, with respect to inventive doublecore-shell particles (bilayer micelle) prepared according to anexemplary embodiment.

FIGS. 2 and 3 are a graph and images of extracted tumors, respectively,for comparing a tumor inhibitory effect of comparative core-shellparticles (monolayer micelles) and inventive double core-shell particles(bilayer micelles) in colorectal cancer mouse models.

FIG. 4 is a graph for comparing a tumor inhibitory effect betweencomparative core-shell particles (monolayer micelles) and inventivedouble core-shell particles (bilayer micelles) in pancreatic cancermouse models (AsPc-1, Xenograft).

FIGS. 5a and 5b are a graph and images of extracted tumors,respectively, for comparing a tumor inhibitory effect of comparativecore-shell particles (monolayer micelles) and inventive doublecore-shell particles (bilayer micelles) in pancreatic cancer mousemodels (MiaPaca-2, Orthotopic).

FIG. 6 is a graph showing the blood concentration of SN-38 Glucuronidefor comparing pharmacokinetic characteristics between comparativecore-shell particles (monolayer micelles) and inventive doublecore-shell particles (bilayer micelles).

DESCRIPTION

The present inventors endeavored to improve the solubility and stabilityof a hydrophobic camptothecin-based compound, of which the applicationis restricted due to severe poor solubility thereof, in spite of stronganticancer activity thereof, and as a result, the present inventorsconfirmed that the mixing of a hydrophobic camptothecin-based compoundand a hydrophilic camptothecin-based compound in an aqueous solvent ororganic solvent leads to a core-shell like particle having core andshell structures formed of hydrophobic- and hydrophilic-basedcamptothecin compounds, respectively. Furthermore, it was confirmed thatthe addition of an amphiphilic block copolymer to the above core-shellparticle forms a shell enclosing the particle. Throughout thedisclosure, this structure that an inner particle formed from acombination of a hydrophobic camptothecin compound and a hydrophiliccamptothecin compound is surrounded by a shell formed of an amphiphilicblock copolymer is described as “double core-shell structure,” “doublecore-shell particle,” or “double micelle,” or “bilayer micelle,” whichare interchangeably used herein.

According to an embodiment of the present invention, the doublecore-shell particle containing both of a hydrophobic camptothecin-basedcompound and a hydrophilic camptothecin-based compound has remarkablyimproved solubility compared with existing poorly soluble camptothecineven without administration in the form of a prodrug. Therefore, thedrug efficacy of active camptothecin (SN-38) can be exerted regardlessof the activity of carboxylesterase II (CESII) in vivo. In addition, itwas confirmed that the particles caused no particle agglomeration orprecipitation even though the particles are again dissolved in anaqueous solvent after freeze-drying, and thus the particles had a dosageform with very excellent stability. Therefore, an aspect of the presentinvention provides a particle composition with a stable doublecore-shell structure, which is advanced from an existing a monolayernanomicelle to resolve a problem of low solubility of a poorly solubledrug.

In accordance with an aspect of the present invention, there is provideda particle comprising: i) a hydrophobic camptothecin-based compound; ii)a hydrophilic camptothecin-based compound; and iii) an amphiphilic blockcopolymer composed of a hydrophobic block and a hydrophilic block.

Herein, camptothecin is a topoisomerase inhibitor found in bark andstalk of the Camptotheca tree (Happy tree) and exhibits a very excellentanticancer effect in the preclinical stage, but cannot be used due tolow solubility thereof. Therefore, many researchers have developedcamptothecin analogues to improve the solubility of camptothecin.Currently, three types of camptothecin derivatives, irinotecan,topotecan, and belotecan, have been approved for cancer chemotherapy.

As used herein, the term “hydrophobicity” refers to the exclusion fromwater molecules and agglomeration, as a tendency shown in non-polarmaterials. When a hydrophobic material is present in a hydrophilicliquid, the hydrophobic material agglomerates while increasinghydrophobic bonds as if the hydrophobic material is afraid of water.

As used herein, the term “hydrophilicity” refers to the property ofbeing dissolved in water with strong affinity with water, as a tendencyshown in polar materials. For example, a hydrophilic polymer compound,or a micelle colloid surface of a surfactant has strong hydrophilicity.

According to an embodiment, the hydrophobic camptothecin-based compoundis selected from the group consisting of 7-ethyl-10-hydroxycamptothecin(SN-38), camptothecin, 10-hydroxycamptothecin, and a pharmaceuticalacceptable salt thereof, but is not limited thereto.

According to another embodiment of the present invention, thehydrophilic camptothecin-based compound is selected from irinotecan,topotecan, belotecan, exatecan, lurtotecan, sinotecan, rubitecan,9-nitrocamptothecin, 9-aminocamptothecin, gimatecan, BNP-1530, DB-67,BN-80915, BN-80927, a pharmaceutically acceptable salt thereof, aglucuronide metabolite thereof, and a glucuronide metabolite of thehydrophobic camptothecin-based compound, but is not limited thereto.

According to a specific embodiment of the present invention, thehydrophobic camptothecin-based compound, which constitutes the innerparticle, may be camptothecin, SN-38, or a mixture thereof, and thehydrophilic camptothecin-based compound, which also constitutes theinner particle together with the hydrophobic camptothecin-basedcompound, may be irinotecan hydrochloride, topotecan hydrochloride, anda glucuronide analog of SN-38.

As used herein, the term “copolymer” refers to a polymer prepared fromtwo or more different types of monomers. For example, the reaction ofstyrene acrylonitrile in a reaction container results in a copolymerhaving both of the two monomers. The term “block copolymer” refers to acopolymer having a form in which a block of one type of monomers islinked to a block of another type of monomers. A case in which a blockof material A is followed by a block of material B is expressed by-[-AB-]-. A chain is called AB type if the chain is composed of only onestrand of each monomer, ABA type if A blocks are present at both ends ofB block in the center, and ABC type if three different types of blocksare present in a main chain. A block copolymer is mainly formed by ionicpolymerization. Unlike other copolymers, this block copolymer has manyphysical properties of a homopolymer formed from two types of monomers.

According to an embodiment of the present invent, the amphiphilic blockcopolymer constituting the particle of the present invention is composedof block A-B or block A-B-A. Here, A is a hydrophilic polymer, which ismonomethoxy polyethylene glycol, dimethoxy polyethylene glycol,polyethylene glycol, polypropylene glycol, monomethoxy polypropyleneglycol, polyethylene oxide, polyacrylic acid, or a polymer thereof, butis not limited thereto. In addition, B is a hydrophobic polymer, whichis polylactic acid, polylactide, polyglycolic acid, polyglycolide, apolylactic acid-co-glycolic acid copolymer, polymandelic acid,polycaprolactone, polydioxan-2-one, polyglutamic acid, polyasparticacid, polyornithine, polyorthoester, a derivative thereof, or acopolymer of two or more compounds selected therefrom, but is notlimited thereto. It would be obvious to a person skilled in the art thatany compound that can constitute an amphiphilic block copolymer usablein the art can be used without limitation.

In a specific embodiment of the present invention, the amphiphilic blockcopolymer is PEG-PCL [poly(ethylene glycol)-b-poly(caprolactone)];PEG-PLA [poly(ethylene glycol)-b-poly(lactic acid)]; mPEG-PGA[monomethoxy poly(ethylene glycol)-b-poly(glycolic acid)]; mPEG-PLGA[monomethoxy poly(ethylene glycol)-b-poly(lactide-co-glycolide)];PEG-PBLA [poly(ethylene glycol)-b-poly(β-benzyl-L-aspartic acid)];PEG-p(Glu) [poly(ethylene glycol)-b-poly(glutamic acid)]; PEG-p(Asp)[poly(ethylene glycol)-b-poly(aspartic acid)]; and/or PEG-PLA-PEG[poly(ethylene glycol)-b-poly(lactic acid)-b-poly(ethylene glycol).

According to an embodiment of the present invention, the hydrophilicpolymer A and the hydrophobic polymer B each has a number averagemolecular weight of 500-10,000 Da, and more specifically 1,000-7,000 Da.When the number average molecular weights of the hydrophilic polymer Aand the hydrophobic polymer B are less than 500 Da or more than 10,000Da, the produced particles have an average size of 200 nm or more andexhibit a multimodal distribution, and thus are difficult to be ananoparticle drug prescribed by the US FDA.

According to another embodiment of the present invention, the weightratio of the hydrophobic camptothecin-based compound and the hydrophiliccamptothecin-based compound, which constitute the particle of thepresent invention, is 1-10:1-10, 1-10:1-5, 1-10:1-3, 1-10:1, 1-5:1-10,1-3:1-10, or 1:1-10, specifically 1-5:1-5, 1-5:1-3, 1-5:1, 1-3:1-5, or1:1-5, and more specifically 1-3:1-3, 1-3:1, or 1:1-3, but is notlimited thereto.

According to an embodiment of the present invention the weight ratio of(a) the sum of the hydrophobic camptothecin-based compound and thehydrophilic camptothecin-based compound and (b) the amphiphilic blockcopolymer is 1:0.1-200, 1:0.5-200 1:1-200, 1:2-200, 1:5-200, 1:10-200,1:50-200, 1:100-200, 1:150-200, 1:0.1-100, 1:0.5-100, 1:1-100, 1:2-100,1:5-100, 1:10-100, 1:20-100, 1:50-100, 1:0.1-50, 1:0.5-50, 1:1-50,1:5-50, 1:10-50, 1:20-50, 1:0.1-20, 1:0.5-20, 1:1-20, 1:5-20, 1:10-20,1:0.1-10, 1:0.5-10, or 1:1-10.

As used herein, the term “to” or “-” between two numerical values meansa section between the numerical values including numerical valuesdescribed before and after the term.

Meanwhile, the particle of the embodiments has a double core-shellstructure comprising the following:

-   -   (a) an inner core-shell containing the hydrophilic        camptothecin-based compound and the hydrophobic        camptothecin-based compound; and (b) an outer core-shell        containing the amphiphilic block copolymer.

In addition, the particle of an embodiment of the present invention hasa bilayer micelle structure.

The amphiphilic block copolymer in which a hydrophilic block and ahydrophobic block are combined at a particular ratio is known to form amicelle through self-assembly in an aqueous solution. The inside of themicelle is hydrophobic, and thus the amphiphilic block copolymer isapplied as a drug delivery system for a poorly soluble preparation. Apolystyrene-polyethylene oxide double block copolymer (PS-b-PEO) is wellknown to form a spherical micelle having an insoluble core (PS) and asoluble shell (PEO) in water.

As described above, the particle of the present invention includes (a) ahydrophilic camptothecin-based compound and a hydrophobiccamptothecin-based compound; and (b) an amphiphilic block copolymer.

As proved in an example of the present invention, the mixing of thehydrophilic camptothecin-based compound and the hydrophobiccamptothecin-based compound significantly increases solubility inaqueous solvents and forms particles. Here, it is likely that thehydrophilic camptothecin-based compound serves as a hydrophilic block ofthe amphiphilic copolymer, and the hydrophobic camptothecin-basedcompound serves as a hydrophobic block of the amphiphilic copolymer,constituting a core-shell structure (micelle).

Therefore, the particle of the present invention forms a double micelle(double core-shell) containing: a monolayer micelle inside; and anamphiphilic block copolymer containing the monolayer micelle inside,wherein in the monolayer micelle, a hydrophilic camptothecin-basedcompound and a hydrophobic camptothecin-based compound constitute acore-shell structure.

More specifically, the hydrophobic block of the amphiphilic blockcopolymer constitutes an insoluble core toward the monolayer micelle,which is relatively hydrophobic and composed of camptothecin-basedcompounds, and the hydrophilic block constitutes a soluble shell towardan external aqueous solvent, and as a result, a double core-shellstructured double micelle is formed.

According to an embodiment of the present invention, the doublecore-shell structured particles spontaneously form particles whendispersed in an aqueous solution.

According to an embodiment of the present invention, the number averageparticle size of the particles is 10-500 nm, 10-400 nm, 10-300 nm, or10-200 nm, and more specifically 20-500 nm, 20-400 nm, 20-300 nm, or20-200 nm. The number average particle size of the particles of theembodiments shows limited change even before or after the particles arefreeze-dried. The reason seems that the hydrophobic and hydrophiliccamptothecin-based compounds primarily constitute an inner core-shellstructured micelle and the amphiphilic block copolymer secondarilyconstitutes an outer shell surrounding the micelle, so that during thefreeze-drying, the secondary outer shell serves as a cryoprotectant thatprevents rapid crystallization, agglomeration, particle collapse of theprimary inner core-shell. As a result, the particles of the embodimentscause no agglomeration or precipitation even when the particles areagain dissolved in the aqueous solvent after freeze-drying. Therefore,the present invention provides a stable double core-shell structuredparticle composition, which is advanced from an existing a monolayernanomicelle to resolve a problem of solubility of a severely poorlysoluble drug.

According to another aspect of the present invention, the presentinvention provides a pharmaceutical composition for treating cancer, thepharmaceutical composition comprising the foregoing particles and apharmaceutically acceptable carrier.

In an embodiment of the present invention, the cancer is selected fromthe group consisting of gastric cancer, ovarian cancer, uterine cancer,small cell lung cancer, non-small cell lung cancer, pancreatic cancer,breast cancer, esophageal cancer, oral cancer, rectal cancer, coloncancer, large intestine cancer, kidney cancer, prostate cancer,melanoma, liver cancer, gall bladder, and other biliary tract cancer,thyroid cancer, bladder cancer, brain and central nervous system cancer,bone cancer, skin cancer, non-Hodgkin's and Hodgkin's lymphoma.

When the particles of embodiments of the present invention or thecomposition containing the same is prepared into a pharmaceuticalcomposition, the pharmaceutical composition of the present invention maycontain a pharmaceutically acceptable carrier. The pharmaceuticallyacceptable carrier is normally used at the time of formulation, andexamples thereof may include, but are not limited to, lactose, dextrose,sucrose, sorbitol, mannitol, starch, acacia gum, calcium phosphate,alginate, gelatin, calcium silicate, microcrystalline cellulose,polyvinylpyrrolidone, cellulose, water, syrup, methylcellulose, methylhydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate, andmineral oil. The pharmaceutical composition of the embodiments mayfurther contain a lubricant, a wetting agent, a sweetening agent, aflavoring agent, an emulsifier, a suspending agent, a preservative, andthe like, in addition to the above ingredients. Suitablepharmaceutically acceptable carriers and agents are described in detailin Remington's Pharmaceutical Sciences (19th ed., 1995).

In a specific embodiment of the present invention, the pharmaceuticalcomposition of the present invention may further contain sucrose,mannitol, sorbitol, glycerin, trehalose, a polyethylene glycol-basedexcipient, and a cyclodextrin-based excipient (alpha-, beta-, andgamma-cyclodextrin, hydroxy cyclodextrin, or a cyclodextrin derivative).The excipient is added to the particles, which correspond to an activeingredient of the present pharmaceutical composition, serves as acryoprotectant or an osmoregulator, and is formulated by freeze-drying,solvent evaporation, or the like.

The pharmaceutical composition of an embodiment of the present inventionmay be administered orally or parenterally, and examples of parenteraladministration may include intravenous administration, subcutaneousadministration, intradermal administration, intramuscularadministration, intranasal administration, mucosal administration,intradural administration, intraperitoneal administration, intraocularadministration, and the like, and specifically, the pharmaceuticalcomposition of the embodiments may be administered intravenously.

The suitable dose of the pharmaceutical composition of an embodiment ofthe present invention varies depending on factors, such as a formulatingmethod, a manner of administration, patient's age, body weight, gender,morbidity, and food, a time of administration, a route ofadministration, an excretion rate, and response sensitivity. Theordinarily skilled practitioners can easily determine and prescribe thedose that is effective for the desired treatment or prevention.According to an embodiment of the present invention, the daily dose ofthe pharmaceutical composition of the present invention is 0.001-100mg/kg.

The pharmaceutical composition of an embodiment of the present inventionmay be formulated into a unit dosage form or may be prepared in amulti-dose container by using a pharmaceutically acceptable carrierand/or excipient according to a method that is easily conducted by aperson having an ordinary skill in the art to which the presentinvention pertains. Here, the dosage form may be a solution in an oilyor aqueous medium, a suspension, an emulsion, an extract, a powder,granules, a tablet, or a capsule, and may further contain a dispersantor a stabilizer.

The pharmaceutical composition of an embodiment of the present inventionmay be administered in parallel with a known compound or pharmaceuticalcomposition having a cancer treatment effect.

In an embodiment of the present invention, the composition furthercontains another type of anticancer drug. Specifically, the compositionof the embodiments further contains a poorly soluble anticancer drug,such as paclitaxel or docetaxel.

The poorly soluble anticancer drugs represented by paclitaxel anddocetaxel have low utilization due to poor solubility, like theabove-mentioned camptothecin, but the poorly soluble anticancer drugshave remarkably improved solubility when contained in the double micelleparticles of an embodiment of the present invention. Therefore, theparticle of an embodiment of the present invention per se is acamptothecin-based anticancer drug, and can be favorably used as a drugdelivery system platform, which can load poorly soluble anticancer drugsor novel candidate drugs with low solubility problems to improvesolubility thereof.

According to still another aspect of the present invention, the presentinvention provides a method for cancer treatment, the method comprisingadministering the foregoing pharmaceutical composition of the presentinvention to a subject in need thereof.

As used herein, the term “administration” or “administer” refers to thedirect application of a therapeutically effective amount of thecomposition to a subject (i.e., an object) with cancer, thereby formingthe same amount thereof in the body of the subject.

The term “therapeutically effective amount” of the composition refers tothe content of the composition, which is sufficient to provide atherapeutic or preventive effect to a subject to be administered, andthus the term has a meaning including “prophylactically effectiveamount.” As used herein, the term “subject” includes, but is not limitedto, human, mouse, rat, guinea pig, dog, cat, horse, cow, pig, monkey,chimpanzee, baboon, or rhesus monkey. Specifically, the subject of thepresent invention is human.

Since the method for cancer treatment of an embodiment the presentinvention includes a step of administering the pharmaceuticalcomposition for cancer treatment according to an aspect of the presentinvention, the overlapping descriptions therebetween are omitted toavoid excessive complication of the specification.

According to another aspect of the present invention, there is provideda method for manufacturing a particle, the method comprising:

-   -   (a) forming an inner core-shell containing a hydrophobic        camptothecin-based compound and a hydrophilic camptothecin-based        compound; and    -   (b) forming an outer core-shell containing an amphiphilic        copolymer.

In an embodiment of the present invention, step (a) comprises mixing thehydrophobic camptothecin-based compound and a hydrophiliccamptothecin-based compound in an organic solvent; and step (b)comprises mixing the inner core-shell and the amphiphilic blockcopolymer in an aqueous solvent.

Since the hydrophobic camptothecin-based compound is severelyhydrophobic, a particular solubilizing technique or preparation methodis needed. In an example of the present invention, in order to overcomesuch poor solubility, a hydrophobic camptothecin-based compound and ahydrophilic camptothecin-based compound were, primarily, simultaneouslydissolved in an organic solvent, and then an organic solvent wascompletely removed by utilizing a rotary vacuum evaporator, therebyobtaining a film form mixture of camptothecin-based compounds. Here, asmall amount of aqueous solvent (e.g., distilled water) was addedthereto with intensive mixing using a vortex-mixer or withultrasonication, thereby obtaining a nano-sized inner core-shellparticle.

Then, an amphiphilic polymer previously dissolved in an aqueous solvent(e.g., distilled water) was added to the inner core-shell particle,followed by homogeneous and strong mixing using a vortex-mixer orultrasonication, thereby obtaining nano-sized double core-shellparticles. Finally, a cryoprotectant and an isotonizing agent was addedthereto to be completely dissolved therein, and then the resultantsolution was filtered through a 0.22-μm sterile filter, followed byfreeze-drying. When added to a 0.9% sodium chloride injection, a 5%glucose injection, or injection water, the final freeze-dried materialis self-assembled to form double core-shell particles with a numberaverage particle size of 20-200 nm.

In the case where the particles manufactured according to the presentinvention have a particle size of 200 nm or less, the non-selectiveremoval of a reticuloendothelial (RES) system in the body can be avoid,and thus it is preferable to manufacture particles having a uniformparticle size of 200 nm or less.

In another embodiment of the present invention, step (a) comprisesmixing a basic aqueous solution, in which a hydrophobic camptothecincompound is dissolved, and an aqueous solution, in which a hydrophiliccamptothecin compound is dissolved; and step (b) comprises mixing theinner core-shell and the amphiphilic block copolymer in an aqueoussolvent to form an amphiphilic block copolymer shell surrounding theinner core-shell.

Here, the aqueous solution in which the hydrophilic camptothecincompound is dissolved may be a basic, neutral, or acidic aqueoussolution.

The inner core-shell can be manufactured by the following method besidesthe manufacturing method in an organic solvent. The hydrophobiccamptothecin-based compound (e.g., camptothecin, SN-38) has very lowsolubility in an acidic or neutral aqueous solution of pH 7 or less, butthe solubility of the hydrophobic camptothecin-based compound is rapidlyincreased in a basic aqueous solution since a lactone ring is opened tohave a form of carboxylic acid. Therefore, the hydrophobiccamptothecin-based compound is dissolved in the basic aqueous solution,and then an aqueous solution in which a hydrophilic camptothecin-basedcompound is dissolved is added thereto to lower the pH to 7 or less, sothat the hydrophobic camptothecin-based compound and the hydrophiliccamptothecin-based compound form a micelle, and the acidity isneutralized and the lactone ring is closed, thereby restoring to theoriginal chemical structure thereof.

In a specific embodiment of the present invention, step (a) comprises:(a1) mixing a basic aqueous solution, in which a hydrophobiccamptothecin-based compound is dissolved, and a basic, neutral, oracidic aqueous solution, in which a hydrophilic camptothecin-basedcompound is dissolved; and (a2) lowering the pH of the mixed aqueoussolution to 7 or lower.

In an example of the present invention, camptothecin or SN-38 isdissolved in a basic aqueous solution, and a hydrophiliccamptothecin-based compound previously dissolved in an aqueous solutionis added to the basic aqueous solution with vigorous mixing usingultrasonication or a vortex-mixer, and an acidic aqueous solution isfurther added thereto to adjust the pH to 7 or less, thereby obtainingcore-shell particles formed from the hydrophobic and the hydrophiliccamptothecin compounds. An amphiphilic polymer previously dissolved inan aqueous solvent (e.g., distilled water) is added to the producedcore-shell particles through vortex-mixing or ultrasonication, therebymanufacturing a double core-shell particle. A cryoprotectant and anisotonizing agent is further added thereto to be dissolved therein,followed by sterile filtration using a 0.22-μm filter, and followed byfreeze-drying.

In the manufacturing method of an embodiment of the present invention,the acidic aqueous solution contains a pharmaceutically acceptableinorganic acid, such as hydrochloric acid, nitric acid, sulfuric acid,or phosphoric acid, and at least one organic acid selected from thegroup consisting of citric acid, malic acid, lactic acid, acetic acid,and tartaric acid. The pH of the acidic aqueous solution is 1-6. Inaddition, the manufacturing method of an embodiment of the presentinvention, the basic aqueous solution contains: an inorganic alkali,including sodium hydroxide, potassium hydroxide, sodiumdihydrogenphosphate, potassium dihydrogenphosphate, magnesium hydroxide,sodium carbonate, and sodium hydrogencarbonate; an alkali salt of anorganic acid; an alkyl amine; or a mixture thereof. The pH of the basicaqueous solution is 8-13.

In still another embodiment of the present invention, step (a) includesmixing the hydrophobic camptothecin-based compound and the hydrophiliccamptothecin-based compound in an organic solvent; and step (b) includesmixing a mixture of the hydrophobic camptothecin-based compound and thehydrophilic camptothecin-based compound with an amphiphilic blockcopolymer in an organic solvent.

In the embodiments, step (a) (manufacturing of inner core-shell) andstep (b) (manufacturing of external amphiphilic copolymer shell) can beperformed in one step.

An example of the present invention shows a method for simultaneouslymanufacturing the inner core-shell and the outer amphiphilic copolymershell. A hydrophobic camptothecin (e.g., 10-hydroxycamptothecin orSN-38), together with a hydrophilic camptothecin (e.g., irinotecanhydrochloride), is added into an organic solvent, and completelydissolved with stirring, and an amphiphilic block copolymer (e.g.,mPEG-PLA) previously dissolved in an organic solvent is added theretowith stirring. The mixture solution was dried by a rotary vacuumevaporator, and an aqueous solvent (e.g., distilled water) is added toresidues, followed by ultrasonication in an ultrasonic cleaner for 10minutes, thereby manufacturing particles in which the hydrophiliccamptothecin and the hydrophobic camptothecin together form the innercore-shell and the amphiphilic block copolymer forms a surroundingshell.

In the manufacturing method of particles, the definitions of ahydrophobic camptothecin-based compound, a hydrophiliccamptothecin-based compound, and an amphiphilic block copolymer, whichconstitute the particles, are as described above with respect to theparticles of embodiments of the present invention.

In the manufacturing method of the present invention, the organicsolvent is a C1-C5 alcohol (methanol, ethanol, propanol, butanol,n-butanol, iso-propanol, 1-pentanol, 2-butoxyethanol, isobutyl alcohol,etc.), an alkyl acetate, acetone, an acetonitrile, chloroform, benzene,toluene, xylene, acetone, a fluoroalkane, pentane, hexane,2,2,4-trimethyl pentane, a decane, a cyclohexane, diisobutylene,1-pentene, 1-chlorobutane, 1-chloropentane, diisopropyl ether,2-chloropropane, 1-chloropropane, chlorobenzene, benzene, diethyl ether,diethyl sulfide, dichloromethane, 1,2-dichloroethane, an aniline, adiethyl amine, an ether, carbon tetrachloride, tetrahydrofuran (THF), ora mixed solvent thereof, but is not limited thereto.

The double core-shell structured particles manufactured by the presentinvention do not precipitate into crystals even when diluted to asufficiently low concentration in an aqueous solution, leading to verystable particles. The particles of the present invention show amono-distribution of particles in an injection solvent before and afterfreeze-drying. The proportion of particles of 200 nm or more is 10% orless, and particles of 500 nm or more are not present. Furthermore, theparticles according to embodiments of the present invention showexcellent results, compared with existing monolayer micelles in animalefficacy tests and pharmacokinetic tests, and does not use a surfactant(Cremophore EL, Pluronic, etc.) causing hypersensitivity, and thus theuse of the particles of the embodiments can provide a pharmaceuticalcomposition or a drug delivery system platform, which are safe for thehuman body.

MODE FOR CARRYING OUT THE INVENTION

Hereinafter, various embodiments of the present invention will bedescribed in detail with reference to examples. These examples are onlyfor illustrating the present invention more specifically, and it will beapparent to those skilled in the art that the scope of the embodimentsis not limited by these examples.

EXAMPLES

Throughout the present specification, the term “%” used to express theconcentration of a specific material, unless otherwise particularlystated, refers to (wt/wt) % for solid/solid, (wt/vol) % forsolid/liquid, and (vol/vol) % for liquid/liquid.

Materials

Among the compounds used in the present invention, hydrophobic andhydrophilic camptothecin-based compounds, polysaccharides such astrehalose, cyclodextrin, polyethylene glycol, and the like were usedfrom Sigma-Aldrich, AbCem, Toronto Research Chemical (Canada) or Tocris(USA), and polymers were used from Akina Inc (USA), Advanced PolymerMaterials (Canada), Shanghai Liang Chemical Co., LTD (China), NanoSoftPolymer (USA), and Samyang BioPharm (Korea).

Example 1 Solubility in Different Solvents, when HydrophobicCamptothecin Compound was Dissolved Alone or Hydrophobic CamptothecinCompound and Hydrophilic Camptothecin Compound were Dissolved in Mixture

The solubility change according to solvent was measured when thehydrophobic camptothecin compound, camptothecin or7-ethyl-10-hydroxy-camptothecin (SN-38) was dissolved alone or togetherwith the hydrophilic camptothecin-based compound, irinotecan (CPT-11),topotecan, or belotecan (CKD-602).

1) Solubility in Different Solvents when Hydrophobic CamptothecinCompound was Dissolved Alone

A supersaturated solution was prepared by adding 20 mg of thehydrophobic camptothecin compound (camptothecin or SN-38) to 5 ml ofethanol, acetonitrile, acetone, ethyl acetate, chloroform dimethylsulfoxide, or distilled water, followed by ultrasonic treatment for 30minutes. The prepared supersaturated solution was filtered through a0.45-μm filter, and a filtrate was properly diluted, followed by HPLCanalysis.

2) Solubility in Different Solvents when Hydrophobic CamptothecinCompound and Hydrophilic Camptothecin Compound were Dissolved in Mixture

The present inventors prepared a supersaturated solution by adding 20 mgof the hydrophilic camptothecin compound (irinotecan hydrochloride,topotecan hydrochloride, or belotecan) to 5 ml of ethanol, acetonitrile,chloroform, ethyl acetate, dimethyl sulfoxide, or distilled water, andthen adding 20 mg of the hydrophobic camptothecin compound (SN-38 orcamptothecin), followed by ultrasonication for 30 minutes. The preparedsupersaturated solution was filtered through a 0.45-μm filter, and afiltrate was properly diluted, followed by HPLC analysis.

HPLC (Agilent 1200 Series, USA) conditions were as follows. The columnwas CapcellPak C8 (5 m, 4.6 mm×25 cm, Shiseido); the mobile phase was amixed solvent of methanol:acetonitrile:buffer (2.8 g/L sodiumdihydrogenphosphate, 1.8 g/L 1-octanesulfonate aqueoussolution)=17:24:59 (v/v); the flow rate was 1.5 mL/min; the measurementwavelength was UV 255 nm; and the sample injection amount was 15 μL. Thesolubility test results are shown in table 1 below.

TABLE 1 Solubility changes of hydrophobic camptothecin-based compoundsin polar organic solvents (unit: mg/ml) Inventive Inventive InventiveInventive Comparative example Comparative example example exampleexample Camptothecin example SN-38 SN-38 SN-38 Camptothecin 20 mg SN-3820 mg 20 mg 20 mg 20 mg Irinotecan 20 mg Irinotecan Topotecan BelotecanSolvent type — 20 mg — 20 mg 20 mg 20 mg Ethanol 0.149 1.414 0.779 2.8852.205 2.047 Acetonitrile 0.101 1.090 0.402 1.972 1.694 1.338 Chloroform0.002 0.051 0.009 0.025 0.088 0.104 Ethyl 0.134 0.295 0.078 0.425 0.2390.221 acetate Dimethyl 3.291 >4 2.263 3. 816 2.949 2.685 sulfoxideDistilled 0.009 0.011 <0.001 <0.001 <0.001 <0.001 water

As shown in Table 1 above, the poorly soluble compound camptothecin orSN-38 alone was hardly dissolved in most solvents including water, andwere dissolved at 2.26-3.29 mg/mL in only dimethyl sulfoxide (DMSO) as anon-volatile solvent. However, camptothecin or SN-38, when dissolvedtogether with the relatively hydrophilic drug, irinotecan hydrochloride,topotecan hydrochloride, or belotecan, had solubility increased up to15-fold, which corresponds to an appropriate level of solubilityrequired for the manufacture of drugs. It was therefore confirmed fromthe above results that the solubility of the hydrophobic camptothecincompounds was remarkably increased when dissolved in mixing with ahydrophilic camptothecin compound.

Example 2 Manufacturing of Core-Shell from HydrophobicCamptothecin-Based Compound and Hydrophilic Camptothecin-Based Compoundin Organic Solvent and Evaluation of Particle Size

In the present example, the hydrophobic camptothecin-based compoundcamptothecin or SN-38, and the hydrophilic camptothecin-based compoundirinotecan hydrochloride, topotecan hydrochloride, belotecan, orSN-38-glucuronide, or an amphiphilic polymer (PEG-PBLA) were dissolvedin an organic solvent to manufacture core-shell particles, and anamphiphilic polymer was added thereto to form a shell surrounding thecore-shell particles.

1) Manufacturing of Core-Shell Particles (Comparative Examples 1 to 6)

The core-shell particles were manufactured by using, as a mainingredient, a hydrophobic camptothecin (camptothecin or SN-38), andwater-soluble camptothecin (irinotecan hydrochloride, topotecanhydrochloride, or SN-38-glucuronide) as an active ingredient or anamphiphilic polymer (poly(ethyleneglycol)-poly(β-butyrolactone-co-lactic acid); PEG-PBLA), as shown in table 2 below.

Specifically, 20 mg of water-soluble camptothecin (irinotecanhydrochloride, topotecan hydrochloride, or SN-38 glucuronide) orPEG-PBLA was added to 20 mg of hydrophobic camptothecin, and 100 ml ofan organic solvent (a 50:50 mixture solution of ethanol andacetonitrile) was added thereto to attain complete dissolution, followedby drying in a rotary vacuum evaporator. 20 ml of distilled water wasadded to the dried product, followed by ultrasonication at 20-30° C. for20 minutes in an ultrasonic cleaner (UC-20, 20 Hz, 400W, Jeio Tech,Korea), thereby obtaining nano-sized particles containing thecamptothecin compound and the PEG-PBLA shell enclosing the camptothecincompound (also referred to as “monolayer micelles” or “comparativecore-shell” herein) in a dispersed state in an aqueous solution. 600 mgof D-trehalose was added to the aqueous solution containing thenano-sized particles, thereby attaining complete dissolution, and thenthe solution was filtered through a 0.22 μm sterile filter, and thefiltrate was freeze-dried. The freeze-drying was conducted for a totalof 62 hours under a temperature cycle of −45° C.→−20° C.→0° C.→20° C. ata vacuum pressure of 100 mTorr or lower, and a freeze-drier from Operon(Korea) was used. An aliquot of the prepared freeze-dried product wastaken, and again dissolved in distilled water for injection, and thesize of particles was measured by the dynamic light scattering (DLS)(Zetasizer™, Malvern, UK).

2) Manufacturing of Inventive Core-Shell Particles (Inventive Examples 1to 4)

In order to prepare a bilayer particle composition having a doublecore-shell structure, the present inventors added 20 mg of each ofhydrophobic camptothecin and hydrophilic camptothecin into 100 ml of anorganic solvent (a 50:50 mixture solution of ethanol and acetonitrile)to be dissolved therein, followed by drying using a rotary vacuumevaporator (Buchi). 200 ml of distilled water was added to the driedproduct, followed by ultrasonication at 20-30° C. for 20 minutes in anultrasonic cleaner, thereby obtaining nano-sized core-shell particlesformed form the mixture of hydrophobic camptothecin and hydrophiliccamptothecin (also referred to as “inner particle” or “inner core-shell”or “inner core” herein) in a dispersed state in an aqueous solution.While the aqueous solution mixed with the inner core-shell particles wasstirred, 90 mg of the amphiphilic block copolymer methoxy poly(ethyleneglycol)-poly(lactide) (mPEG-PLA) (mPEG molecular weight:PLA molecularweight=2,000:1,500) previously dissolved in 10 ml of distilled water wasslowly added, followed by stirring at 20-30° C. for 6 hours, therebypreparing double core-shell particles having a structure that anamphiphilic block copolymer shell surrounds the inner core-shell (alsoreferred to as “double core-shell,” “double core-shell particle,” or“bilayer micelle” herein), in a dispersed state in an aqueous solution.600 mg of D-trehalose as a cryoprotectant was added to the aqueoussolution containing double core-shell particles to be dissolved therein,and then the mixture was filtered through a 0.22-μm sterile filter, andthen freeze-drying was conducted by the same method as in themanufacturing of primary core-shell particles, thereby obtaining afreeze-dried product as a white powder. A predetermined amount of thefreeze-dried product was again dissolved in injection water to measurethe size of the particles. In addition, the average particle sizesafter/before freeze-drying were measured, and the proportion of particlewith 200 nm or more and the distribution pattern (mono- or multi-modaldistribution) were compared.

TABLE 2 Proportions in manufacturing of particles (unit: mg) Poorlysoluble camptothecin Water-soluble camptothecin Micelle SN- IrinotecanTopotecan SN-38 PEG-PBLA mPEG-PLA type Camptothecin 38 hydrochloridehydrochloride glucuronide (5k:6.5k) (2k:1.5k) Comparative Mono- 20 — 20— — — — example 1 Comparative Mono- 20 — — — 20 — — example 2 InventiveBi- 20 — 20 — — — 90 example 1 Comparative Mono- — 20 20 — — — — example3 Comparative Mono- — 20 — 20 — — — example 4 Comparative Mono- — 20 — —20 — — example 5 Comparative Mono- — 20 — — — 20 — example 6 InventiveBi- — 20 20 — — — 90 example 2 Inventive Bi- — 20 — 20 — — 90 example 3Inventive Bi- — 20 — — 20 — 90 example 4 *PEG-PBLA: poly(ethyleneglycol)-b-poly(3-benzyl-L-aspartic acid) *mPEG-PLA: methoxypoly(ethyleneglycol)-b-poly(lactic acid)

TABLE 3 Comparision results of particle size and distributionbefore/after freeze-drying in particles Particle size before freeze-drying (n = 3) Average Particle size after freeze-drying (n = 3)particle >200 nm Average >200 nm Micelle size Propotion particleproportion Pariticle type (nm) (%) size (nm) (%) >1 μm distributionComparative Mono- 123.9 5.9 156.1 40.1 O Multi- example 1 ComparativeMono- 133.6 7.1 152.8 38.6 O Multi- example 2 Inventive Bi- 135.8 3.6148.9 4.8 ND Mono- example 1 Comparative Mono- 76.2 0.0 108.4 16.4 OMulti- example 3 Comparative Mono- 78.5 0.1 115.4 28.5 O Multi- example4 Comparative Mono- 92.1 2.7 131.2 30.4 O Multi- example 5 ComparativeMono- 102.8 5.9 138.4 34.2 O Multi- example 6 Inventive Bi- 83.3 0.093.8 2.4 ND Mono- example 2 Inventive Bi- 88.2 0.1 99.8 2.8 ND Mono-example 3 Inventive Bi- 93.3 0.2 105.4 3.1 ND Mono- example 4 *ND: Notdetected

As shown in Table 3 above, as for the average particle size afterfreeze-drying, the comparative core-shell particles (monolayer micelles)composed of poorly soluble camptothecin and water-soluble camptothecinor the primary core-shell particles composed of poorly solublecamptothecin and an amphiphilic polymer were increased to about 1.2- to1.5-fold, but the inventive double-core-shell particles (bilayermicelles) only increased about 1.1-fold.

With respect to the comparative core-shell particles (monolayermicelles), after the freeze-drying, the particles of 200 nm or more wereproduced in large amounts, about 16-40%, and especially, the particlesof several micrometers (μm) or more, capable of influencing safety whenadministered to the human body, were produced (comparative examples 1 to6 on table 3 and FIGS. 1a and 1b ). Whereas, with respect to inventivedouble core-shell particles, the particles of 200 nm or more weredetected in about 2.4-3.1% for SN-38 and about 4.8% for camptothecin,both being less than 5%, showing very favorable results, and theparticles of 500 nm or more were not observed (Inventive examples 1 to 4in table 3, and FIG. 1c ).

In the particular distribution, the comparative core-shell particles(monolayer micelles) showed a distribution with multiple peaks (FIGS. 1aand 1b ), but the inventive core-shell particles showed amono-distribution, confirming a very stable structure (FIG. 1c ). Inaddition, the average particle size of the comparative core-shellparticles (monolayer micelles) was smaller than that of the inventivedouble core-shell particles (bilayer micelles) by about 10 nm beforefreeze-drying, but the change of the particle size before and afterfreeze-drying was very great, whereas the inventive double core-shellparticles (bilayer micelles) were very physically stable.

Example 3 Evaluation of Stability of Comparative Core-Shell Particlesand Inventive Double Core-Shell Particles

In the present example, monolayer micelles (comparative examples 1, 3,and 6) and double core-shell particles (inventive examples 1 and 2) werecompared for the change in particle size over the time. Sample productsmanufactured according to the drug stability test standards were storedfor six months in accelerated test conditions (40□, 75% relativehumidity). The results are shown in Table 4.

TABLE 4 Test results of stability of Comparative core-shell particles(monolayer micelles) and Inventive double core-shell particles (bilayermicelles) 0 month 3 months 6 months Average Average Averageparticle >200 nm particle >200 nm particle >200 nm Micelle sizePropotion size Propotion size Propotion type (nm) (%) (nm) (%) (nm) (%)Comparative Mono- 156.1 40.1 172.1 47.6 196.6 58.1 example 1 InventiveBi- 148.9 4.8 155.0 5.6 168.3 7.4 example 1 Comparative Mono- 108.4 16.4126.7 28.1 151.9 31.6 example 3 Comparative Mono- 138.4 34.2 149.9 44.6174.2 48.4 example 6 Inventive Bi- 93.8 2.4 99.5 3.5 112.9 3.8 example 2

The comparative core-shell particles (monolayer micelles), containingcamptothecin or SN-38 as a main ingredient, and inventive doublecore-shell particles (bilayer micelles) were subjected to stabilitytests. As a result, in the case of the monolayer micelles, the averageparticle size was increased by about 50% or more and the proportion ofthe particles of 200 nm or more was rapidly increased to 58% inaccelerated test conditions. In the case of the bilayer particles, theaverage particle size was increased by about 20%, and the proportion ofthe particles of 200 nm or more was restricted within 3.8% for SN-38 and7.4% for camptothecin. Therefore, it can be seen that the structure ofthe inventive particles according to embodiments of the presentinvention was significantly improved in view of stability, compared withthe comparative particles.

Example 5 Manufacturing of Double Core-Shell Particles According to Typeof Amphiphilic Polymer and Molecular Weight of Amphiphilic Polymer

In the present example, inventive double core-shell particles weremanufactured using various amphiphilic polymers (block copolymers). Asshown in Table 5, 20 mg of each of SN-38 and irinotecan hydrochloride ashydrophobic and hydrophilic camptothecin compounds, 500 mg of trehaloseas a cryoprotectant, and 90 mg of each of amphiphilic polymers wereused. The manufacturing method was carried out in the same manner as inthe core-shell particle manufacturing method in example 2 above, and thematerials were again dissolved in injection water after freeze-drying tocompare particular sizes.

TABLE 5 Particle size comparision of bilayer particles afterfreeze-drying according to amphiphilic polymer type and averagemolecular weight thereof Polymer type >1 μm Particle (AverageParticle >200 nm presence distribution molecular size Proportion or(Mono-/ No weight) (nm) (%) absence Multi-) PDI Inventive PEG-PCL 133.85.0 ND Mono- 0.271 example 5 (5k:2.5k) Inventive PEG- 102.0 3.1 ND Mono-0.209 example 6 PCL(2k:1.5k) Inventive PEG- 99.2 2.9 ND Mono- 0.225example 7 PLA(2.5k:1k) Inventive mPEG- 99.7 2.6 ND Mono- 0.213 example 8PGA(2k:1.5k) Inventive mPEG- 101.5 3.4 ND Mono- 0.247 example 9PLGA(1k:1k) Inventive PEG- 137.9 9.8 ND Mono- 0.287 examplePBLA(5k:6.5k) 10 Inventive PEG- 122.1 6.5 ND Mono- 0.245 example p(Glu)(5k:2.5k) 11 Inventive mPEG- 128.2 7.6 ND Mono- 0.231 example p(Asp)(5k:2.5k) 12 Inventive PEG-PLA-PEG 156.4 11.8 ND Mono- 0.298 example(2.5k-1k-2.5k) 13 *PEG-PCL: poly(ethylene glycol)-b-poly(caprolactone)*PEG-PLA: poly(ethylene glycol)-b-poly(lactic acid) *mPEG-PGA:monomethoxy poly(ethylene glycol)-b-poly(glycolic acid) *mPEG-PLGA:monomethoxy poly(ethylene glycol)-b-poly(lactide-co-glycolide)*PEG-PBLA: poly(ethylene glycol)-b-poly(β-benzyl-L-aspartic acid)*PEG-p(Glu): poly(ethylene glycol)-b-poly(glutamic acid) *PEG-p(Asp):poly(ethylene glycol)-b-poly(aspartic acid) *PEG-PLA-PEG: poly(ethyleneglycol)-b-poly(lactic acid)-b-poly(ethylene glycol) *PDI: Polydiversityindex

As shown in Table 5 above, it can be seen that the inventive particlesof embodiments of the present invention can be manufactured by usingvarious amphiphilic polymers (block copolymers) and amphiphilic polymershaving various average molecular weights, and the stability of theparticles was excellent.

Example 6 Evaluation of Particle Size According to Type ofCryoprotectant

In the present example, the effects of a cryoprotectant in themanufacturing of freeze-dried double core-shell particles were observed.Here, 10-hydroxycamptothecin and SN-48 were selected as hydrophobiccamptothecin compounds; irinotecan hydrochloride was selected as ahydrophilic camptothecin; and mPEG-PLA (2 k:1.5 k) was used as anamphiphilic polymer. As a cryoprotectant, 500 mg of each of D-trehalose,D-mannitol, PEG2000, and hydroxypropyl-β-cyclodextrin (HP-b-CD) wasused. The double core-shell particles were manufactured using thecompositions shown in Table 6, and the manufacturing method was carriedout in the same manner as in example 2.

TABLE 6 Size of double core-shell particles according to cryoprotectanttype 10-OH >200 nm >1 μm Camptothecin SN-38 Irinotecan mPEG-Cryoprotectant Particle Proporiton presence (mg) (mg) (mg) PLA (500 mg)size (nm) (%) or absence Inventive 20 — 20 90 Mannitol 121.8 4.4 NDexample 13 Inventive — 20 20 90 Mannitol 101.1 2.9 ND example 14Inventive 20 — 20 90 Trehalose 118.6 5.8 ND example 15 Inventive — 20 2090 Trehalose 99.6 3.1 ND example 16 Inventive — 20 20 90 PEG2000 133.219.6 O example 17 Inventive — 20 20 — PEG2000 164.9 32.8 O example 18Inventive — 20 20 90 HP-b-CD 126.8 9.3 O example 19 Inventive — 20 20 —HP-b-CD 151.7 26.2 O example 20 *PEG2000: Polyethyleneglycol 2000*HP-b-CD: hydroxypropyl-β-cyclodextrin *ND: Not detected

As shown in Table 6, favorable results were observed in view of theparticle size when the polysaccharides mannitol and trehalose were usedas cryoprotectants. Whereas, the particle sizes were somewhat large, forexample, particles with a size of 1 μm or more were detected, in PEG2000and HP-b-CD. In inventive examples 18 and 20 for monolayer micelles,relatively large particle of 200 nm or more and macroparticles of 1 μmor more were observed when polyethylene glycol and cyclodextrin wereused as cryoprotectants.

Example 7 Manufacturing of Inner Core-Shell Particles and DoubleCore-Shell Particles in Water-Soluble Solvent Conditions

The inner core-shell particles can be manufactured in an aqueoussolution as well as an organic solvent as in example 2. As shown inTable 7 below, 10 mg of SN-38 was completely dissolved in 0.1 ml of a0.5 M sodium hydroxide aqueous solution, and the resultant solution wasdropped and neutralized in an aqueous solution of irinotecanhydrochloride (1 mg/ml) previously dissolved in 15 ml of a 0.5 mMhydrochloride aqueous solution, and a hydrochloride aqueous solution wasfurther added to control the pH to about 5, followed by ultrasonication,thereby obtaining inner core-shell particles. 40 mg of the amphiphilicpolymer mPEG-PLA was added thereto, followed by stirring at roomtemperature for 6 hours, and 300 mg of D-trehalose was further added tobe dissolved. The mixture solution was filtered through a 0.22-μmsterile filter, freeze-dried, and again dissolved in injection water,and then the particle size was measured (inventive example 21). SN-38and irinotecan were dissolved using the organic alkali ethanol amine asa basic aqueous solution or the organic acid citric acid as an acidicaqueous solution, and bilayer particles were manufactured by the samemethod, thereby measuring the particle sizes, respectively (inventiveexamples 22 and 23).

TABLE 7 Manufacturing of bilayer particles under basic and acidicaqueous solutions Type of acid and basic solvents and particle size (nm)SN- NaOH/ 38 Trinotecan mPEG-PLA NaOH/ Ethanolamine/ Citric (mg) (mg)Trehalose (2k:1.5k) HCl HCl acid Inventive 10 10 300 40 120.1 — —example 21 Inventive 10 10 300 40 — 122.3 — example 22 Inventive 10 10300 40 — — 119.5 example 23

As shown in Table 7, the double core-shell particles manufactured bydissolving hydrophobic camptothecin and hydrophilic camptothecin inbasic and acidic aqueous solutions showed a particle size of about 120nm, and thus the double core-shell particles were successfullymanufactured.

Example 8 Mixing Manufacturing of Double Core-Shell Particles

The present example showed a method for manufacturing double core-shellparticles by mixing all the hydrophobic camptothecin, hydrophiliccamptothecin, and amphiphilic block copolymer in one step. Hydrophobiccamptothecin compounds (10-hydroxycamptothecin and SN-38) were placedtogether with 20 mg of irinotecan hydrochloride in 100 ml of an organicsolvent (50:50 mixture solution of ethanol:acetonitrile), and completelydissolved with stirring, and 90 mg of mPEG-PLA (2 k:1.5 k) dissolved in10 ml of an organic solvent (50:50 mixture solution ofethanol:acetonitrile) was added thereto with stirring. The mixturesolution was dried by a rotary vacuum evaporator, and 200 ml ofdistilled water was added to residues, followed by ultrasonication for10 minutes in an a ultrasonic cleaner, thereby obtaining doublecore-shell particles of the embodiments. 400 mg of D-mannitol as acryoprotectant was added to be dissolved, and this solution was filteredthrough a 0.22-μm sterile filter, freeze-dried. A proper amount of thefreeze-dried product was again dissolved in injection water to measurethe particle size. The results are shown in Table 8.

TABLE 8 Particle size of double core-shell particles (bilayer micelles)produced by mixing manufacturing >1 μm SN- 10-OH Irino- mPEG- D-Particle >200 nm Presence 38 Camptothecin tecan PLA mannitol sizeProportion or (mg) (mg) (mg) (mg) (mg) (nm) (%) absence Inventive 20 —20 90 400 125.4 4.2 ND example 24 Inventive — 20 20 90 400 129.3 4.8 NDexample 25 *ND: Not detected

As shown in Table 8, the double core-shell particles manufactured bysimultaneously mixing and dissolving hydrophobic camptothecin,hydrophilic camptothecin, and amphiphilic polymer in an organic solventshowed a mono-distribution of particle sizes of about 120-130 nm,wherein particles of 200 nm or more were detected in small amounts, 5%or less, but particles of 1 μm or more were not detected, and thus theparticles were confirmed to have overall favorable stability.

Example 9 Tumor Inhibitory Effect Comparison Test of ComparativeCore-Shell Particles and Inventive Double Core-Shell Particles in TumorMouse Models (Colorectal Cancer)

In colorectal mouse models, compositions comprising the monolayermicelle of comparative examples 3 and 6 and compositions comprisingbilayer particles of inventive example 2 were measured for anticancereffect by the following method.

The previously cultured colorectal cancer cell line (HT-29) was injectedat 5×10⁶ cells/0.2 mL into the right flank of Balb/c nude mice, andafter about 7 days, only tumors with a size of 150-200 mm³ wereselected. Nine animals were assigned to each group, and wereintravenously administered with a sham drug (non-treatment group),comparative example 3 (monolayer micelles), comparative example 6(monolayer micelles), and inventive example (bilayer particles), onceevery three days, three times in total. The dose was 10 mg/kg on thebasis of SN-38. The volume of tumor measured every three days after theadministration of the test composition was used as a measurement indexof the anticancer effect, and was observed for a total of 18 days. Theresults are shown in FIGS. 2 and 3.

As a result of measurement of tumor inhibitory effect, the monolayermicelle compositions (comparative examples 3 and 6) showed a tumorinhibitory effect of about 50-60% compared with a negative controlgroup, and the bilayer particle composition (inventive example 2) showeda tumor inhibitory effect of about 80% or more compared with a negativecontrol group, indicating very excellent effects. These results were dueto the fact that the stabilized micelle structure and the micellestructure having a size as small as 200 nm or less of the embodimentswere efficiently transferred to cancer tissues while stably staying inthe body.

Example 10 Tumor Inhibitory Effect Comparison Test of ComparativeCore-Shell Particle and Inventive Bilayer Particles in Pancreatic CancerMouse Models (AsPc-1)

The tumor inhibitory effect of a composition comprising the monolayermicelle (comparative core-shell particle) of comparative example 3 wascompared with that of a composition comprising the bilayer particles ofinventive example 2) in pancreatic cancer mouse models. The previouslycultured pancreatic cancer cell line (AsPc-1) was injected at 5×10⁶cells/0.2 mL into the right flank of male BALB/c-nu/nu mice, and afterabout 10 days, only tumors with a size of 100-150 mm³ were selected. Tenanimals were assigned to each group, and were administered with a shamdrug (non-treatment group), comparative example 3, and inventive example2, once every seven days, three times in total. The dose was 10 mg/kg onthe basis of SN-38. The volume of tumor measured every three days afterthe administration of the test composition was used as a measurementindex of the anticancer effect, and was observed for a total of 24 days.The results are shown FIG. 4.

As a result of measurement of tumor inhibitory effect, the monolayermicelle composition (comparative example 3) showed a tumor inhibitoryeffect of about 27% compared with a negative control group, and thebilayer particle composition (inventive example 2) showed a tumorinhibitory effect of about 47% or more compared with a negative controlgroup, indicating very excellent effects. The results were overallsimilar to those in the colorectal cancer models (Example 9), and it wasconfirmed that the bilayer particle composition of the embodiments werevery excellent in tumor inhibitory effects compared with monolayermicelles.

Example 11 Tumor Inhibitory Effect Comparison Test of ComparativeCore-Shell Particle Composition and Inventive Bilayer ParticleComposition in Pancreatic Cancer Mouse Models (MiaPaca-2)

The tumor inhibitory effect of a composition comprising the monolayermicelle (comparative core-shell particle) of comparative example 3 wascompared with that of the bilayer particle composition (inventiveexample 2) in pancreatic cancer mouse models (Orthotopic) on the basisof SN-38. After the left flank side of male BALB/c-nu/nu mice wasincised at 0.7-1 cm, the entire pancreas and spleen were exposed to theoutside, and then the pancreatic cancer cell line (MiaPaca-2 cell line)previously cultured using a syringe was injected at 1×10⁷ cells/0.1 mL.It was confirmed that the tumor cell suspension did not leak, and theorgans exposed to the outside were relocated again, and the incisionsite was sutured by suture thread. About 10 days after the inoculationof the pancreatic cancer cell line, grouping was carried out by the bodyweight. Ten animals were assigned to each group, and were administeredwith a sham drug (non-treatment group), comparative example 3, and testexample 2, once every seven days, three times in total. The dose was 20mg/kg on the basis of SN-38. The animals were observed for a total of 28days, and the tumor size and weight were measured by autopsy on day 28.The results are shown FIG. 5.

As a result of measurement of tumor inhibitory effect, the tumor weightof the non-treatment group (negative control group) was on average0.46±0.17 g, the tumor weight of the monolayer micelle composition(comparative example 3) treatment group was 0.37±0.09 g, and the tumorweight of the bilayer particle composition (inventive example 2)treatment group was 0.21±0.05 g. Therefore, the bilayer particlecomposition showed a tumor inhibitory effect of 55% or more comparedwith the monolayer micelle, and thus a very excellent tumor inhibitoryeffect.

Example 12 Pharmacokinetic Test in Beagle Dogs

The pharmacokinetic characteristics of the monolayer micelle composition(comparative example 3) were compared with those of the bilayer particlecomposition (inventive example 2) in beagle dogs. Male beagle dogsweighing 7-10 kg were divided into two groups, three dogs per eachgroup, according to the body weight, and were intravenously administeredwith a composition comprising the monolayer micelle (comparativecore-shell particle) of comparative example 2 and a bilayer particlecomposition (inventive example 2) at 0.5 mg/kg on the basis of SN-38 for10 minutes infusion. Blood samples were taken at 0.33, 0.67, 1, 1.5, 2,4, 8, 12, 24, 36 hours after the end of the administration, and theplasma obtained by centrifuging the blood was pretreated by thefollowing method to measure the drug concentration in plasma. For samplepretreatment, 20 μL of S-(+)-camptothecin (500 ng/mL, dissolved inacetonitrile) as an internal standard substance was first added to 100μL of plasma, and 500 μL of acetonitrile was further added, followed byvortex-mixing for 30 seconds. After the mixture was centrifuged at12,000 rpm for 3 minutes, the supernatant was taken, and was injected 2μL into an LC-MS/MS system (API-5,000 model, AB Sciex). Separation wascarried out while the column was Gemini C18 (3 μm, 2.0×50 mm,Phenomenex, USA), the mobile phase was a 50% acetonitrile solutioncontaining 0.1% formic acid, and the flow rate was 0.25 mL/min. MS/MSdetection conditions were positive ion mode, and SN-38 glucuronide wasdetected at m/z 569.3→393.2, and the internal standard was detected atm/z 349.2→305.2.

The blood drug concentration over time after administration is shown inFIG. 6, and pharmacokinetic parameters therefor are shown in Table 9.

TABLE 9 Pharmacokinetic parameter comparison between monolayer micelleparticle composition and bilayer particle composition Micelle Dose CmaxAUCt Relative No. type (mg/kg) (ng/mL) Tmax(hr) (ng · hr/mL) BA(%)Comparative Monolayer 0.5 25.30 ± 5.12 0.33 ± 0.00 131.84 ± 34.94 —example 3 micelles Inventive Double 0.5 66.25 ± 16.34 0.44 ± 0.20 361.29± 96.25 275.6 example 2 core- shell *mean ± SD (n = 3)

In beagle dogs, SN-38 glucuronide produced directly from SN-38solubilized in the particles of the embodiments was analyzed. Theresults confirmed that the bilayer particles of the embodiments of thepresent invention showed an increase in bioavailability by about 2.75times, compared with the comparative monolayer micelles. It wasdetermined from the above results that the inventive bilayer particlesmaximize the solubility of the poorly soluble drug SN-38 in vivo.

Although the present invention has been described in detail withreference to the specific features, it will be apparent to those skilledin the art that this description is only for a preferred embodiment anddoes not limit the scope of the present invention.

1. A method for treating a subject with a cancer, comprisingadministering a therapeutically effective amount of a composition to thesubject, wherein the composition comprises freeze-dried particles withparticle size of less than 1 μm, said freeze-dried particles comprising(a) an inner core comprising (i) a hydrophobic camptothecin compound;and (ii) a hydrophilic camptothecin-based compound; and (b) an outershell surrounding the inner core, said outer shell being formed of anamphiphilic block copolymer, wherein the amphiphilic block copolymercomprises a hydrophobic block and a hydrophilic block in a same chain;wherein the hydrophobic camptothecin compound is selected from the groupconsisting of 7-ethyl-10-hydroxycamptothecin (SN-38), camptothecin,10-hydroxycamptothecin, a pharmaceutical acceptable salt thereof, and acombination thereof; wherein the hydrophilic camptothecin compound isselected from irinotecan, topotecan, belotecan, exatecan, lurtotecan,sinotecan, rubitecan, 9-nitrocamptothecin, 9-aminocamptothecin,gimatecan, BNP-1530, DB-67, BN-80915, BN-80927, a pharmaceuticallyacceptable salt thereof, a glucuronide metabolite thereof, and aglucuronide metabolite of the hydrophobic camptothecin compound, and acombination thereof; and wherein a proportion of the freeze-driedparticles with a size more than 200 nm is 11.8% or less as measured bydynamic light scattering (DLS) method.
 2. The method of claim 1, whereinthe cancer is selected from the group consisting of gastric cancer,ovarian cancer, uterine cancer, small cell lung cancer, non-small celllung cancer, pancreatic cancer, breast cancer, esophageal cancer, oralcancer, rectal cancer, colon cancer, large intestine cancer, kidneycancer, prostate cancer, melanoma, liver cancer, gall bladder and otherbiliary tract cancer, thyroid cancer, bladder cancer, brain and centralnervous system cancer, bone cancer, skin cancer, non-Hodgkin's andHodgkin's lymphoma, and blood cancer.
 3. The method of claim 1, whichfurther comprises administering an additional anti-cancer drug to thesubject.
 4. The method of claim 3, wherein the additional anti-cancerdrug is administered simultaneously or sequentially with thecomposition.
 5. The method of claim 1, wherein the composition furthercomprises an additional anti-cancer drug.
 6. The method of claim 5,wherein the additional anti-cancer drug is poorly soluble anti-cancerdrug.
 7. The method of claim 6, wherein the poorly soluble anti-cancerdrug is paclitaxel or docetaxel.
 8. The method of claim 6, wherein thepoorly soluble anti-cancer drug is contained in the inner core.
 9. Themethod of claim 1, wherein the administering of the composition reducesa volume of cancerous tissue in the subject.
 10. The method of claim 1,wherein the composition is administered intravenously, subcutaneously,intradermally, intramuscularly, intranasally, mucosally, intradurally,intraperitoneally, or intraocularly.
 11. The method of claim 1, whereinthe composition is a reconstituted solution of the freeze-driedparticles dissolved in a solvent.
 12. The method of claim 1, wherein theamphiphilic block copolymer comprises an A-B block or A-B-A block,wherein A is a hydrophilic segment formed from monomethoxy polyethyleneglycol, dimethoxy polyethylene glycol, polyethylene glycol,polypropylene glycol, monomethoxy polypropylene glycol, polyethyleneoxide, polyacrylic acid, or a polymer thereof; and wherein B is ahydrophobic segment formed from polylactic acid, polylactide,polyglycolic acid, polyglycolide, a polylactic acid-co-glycolic acidcopolymer, polymandelic acid, polycaprolactone, polydioxan-2-one,polyglutamic acid, polyaspartic acid, polyornithine, polyorthoester, ora copolymer thereof.
 13. The method of claim 1, wherein the hydrophobiccamptothecin compound is 7-ethyl-10-hydroxylcamptothecin (SN-38),wherein the hydrophilic camptothecin compound is irinotecan, topotecan,or SN-38 glucuronide, and wherein the amphiphilic block copolymer isselected from the group consisting of PEG-PBLA, mPEG-PLA, PEG-PCL,PEG-PLA, mPEG-PGA, mPEG-PLGA, PEG-p(Glu), PEG-PLA-PEG, and PEG-p(Asp).14. The method of claim 1, wherein a weight ratio of the hydrophobiccamptothecin compound and the hydrophilic camptothecin compound is 1:10to 10:1.
 15. The method of claim 1, wherein a weight ratio of a total ofthe hydrophobic camptothecin compound plus the hydrophilic camptothecincompound and the amphiphilic block copolymer is 1:200 to 10:1.
 16. Themethod of claim 1, wherein the hydrophobic camptothecin compound, thehydrophilic camptothecin compound, and the amphiphilic block copolymerare respectively: i) SN-38, irinotecan, and an amphiphilic blockcopolymer selected from the group consisting of PEG-PBLA, mPEG-PLA,PEG-PCL, PEG-PLA, mPEG-PGA, mPEG-PLGA, PEG-p(Glu), PEG-PLA-PEG, andPEG-p(Asp); or ii) SN-38, topotecan, and mPEG-PLA.
 17. The method ofclaim 1, wherein the number average particle size of the freeze-driedparticles is 10-500 nm.
 18. The method of claim 1, wherein the numberaverage particle size of the freeze-dried particles is 20-200 nm. 19.The method of claim 1, wherein the composition further comprises acryoprotectant.
 20. The method of claim 19, wherein the cryoprotectantis mannitol or trehalose.